Identification of Proteolytic Thermophiles from Moinit Coastal Hot-Spring, North Sulawesi, Indonesia

We have isolated thermophilic bacteria from Moinit coastal hot-spring, North Sulawesi, Indonesia. Of 12 seawater samples collected, we obtained 20 thermophilic bacterial isolates. Sixteen of the isolated thermophiles were found to exhibit protein-degrading ability as indicated by the presence of clearing zones surrounding their colonies on skim milk-containing plates at 65 degrees C. Five of them with larger proteolytic indexes, designated as 2b, 4b, 9a, 9b, and 12L, were selected for further studies. The ability of these five strains to hydrolyze casein at high temperatures was indicated by relatively high activity of their extracellular proteases at 60 degrees C. Partial protease-encoding genes were amplified from each isolated strain and sequenced. Further bioinformatics analysis suggests that the extracellular proteases that they produced belong to serine proteases. The integrated morphological, biochemical, and genetic analyses showed the identity of the strains 2b, 4b, and 9a as members of Bacillus, 9b as Pseudomonas, and 12L as Aneurinibacillus. To the best of our knowledge, so far thermophilic Pseudomonas and Aneurinibacillus remain poorly investigated in term of extracellular protease production, suggesting the biotechnological potential of the strains 9b and 12L as the producer of a unique extracellular thermostable protease.